👤 Muhammad Yousef

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide, with tumor progression often driven by dysregulated oncogenic pathways. USP6NL, a known regulator of endocytic trafficking, has recently been implicated in tumorigenesis. However, its precise role in CRC remains unclear, and more studies are still needed to deepen our understanding of underlying mechanisms implicated in its oncogenic role. Therefore, silencing USP6NL could provide a novel therapeutic strategy by concurrently disrupting several oncogenic mechanisms, creating a new avenue for CRC management, particularly in patients who develop resistance against conventional therapies. This study investigates the impact of USP6NL knockdown on CRC cell morphology, proliferation, apoptosis, migration, angiogenesis, and metabolic adaptation, providing mechanistic insights into its oncogenic functions. HCT116 colorectal cancer cells were transfected with USP6NL-specific siRNA. Immunocytochemistry was used to confirm successful silencing, functional assays were performed to assess changes in cell morphology using phase-contrast and scanning electron microscopy, and colony formation and wound healing assays were performed to assess cell clonogenic capacity and migration, respectively, in addition to apoptosis assay via flow cytometry, and RT²-Profiler PCR array to measure variation in gene expression of 84 cancer-related genes. Statistical analyses were performed to evaluate significant differences between control and USP6NL-silenced groups. USP6NL depletion led to profound morphological changes, including membrane blebbing, cell shrinkage, and loss of adhesion, reflecting late apoptotic features of cells. These findings were further supported by flow cytometry, which confirmed increased apoptosis, with a higher proportion of late apoptotic cells (20.99% in USP6NL knockdown vs. 2.69% in control, p = 0.042). Colony formation assays revealed a significant reduction in the clonogenic potential, suggesting a critical role of USP6NL in promoting CRC cell proliferation (p ≤ 0.05). The wound healing assay demonstrated impaired migration in USP6NL-silenced cells, with a marked delay in wound closure (p = 0.0201 at 48 h). Gene expression analysis revealed a significant downregulation of VEGFC (-8.62-fold) and ANGPT2 (-4.03-fold), impairing angiogenesis and suppressing FOXC2, SNAI1, and SNAI2, indicating EMT inhibition. Additionally, CASP9, APAF1, and BCL2L11 were upregulated, confirming the activation of intrinsic apoptosis, while metabolic regulators HIF1A and LDHA were downregulated, suggesting impaired tumor hypoxic adaptation. This study establishes USP6NL as a key modulator of CRC progression, regulating proliferation, apoptosis, migration, angiogenesis, and metabolic pathways. The loss of USP6NL leads to EMT suppression, apoptosis induction, and reduced tumor cell viability, positioning it as a potential therapeutic target in colorectal cancer. Further investigations are warranted to explore USP6NL's interactions in oncogenic signaling networks and its feasibility as a target for CRC therapy. It could serve as a promising therapeutic target in colorectal cancer, potentially enhancing tumor cell death and limiting metastasis. Targeting USP6NL could also provide a novel approach in combination with existing therapies, improving treatment efficacy and reducing side effects. Show less

Prefoldin1 (PFDN1), a molecular chaperone, is essential for stabilizing cytoskeletal proteins like actin and tubulin, supporting cellular processes such as survival, migration, and cell cycling. Recent evidence suggests that PFDN1 also influences key cancer-related signaling pathways. However, the complete mechanisms involved and the downstream genes implicated in such action remain relatively undiscovered. This study investigated the effects of PFDN1 silencing on cellular processes and gene expression in triple-negative breast cancer (TNBC) cells, focusing on its potential as a therapeutic target. MDA-MB-231 cells, a TNBC model, were transfected with PFDN1-targeting siRNA to knock down PFDN1 expression. The effects of PFDN1 silencing were assessed through various assays, including phase contrast and scanning electron microscopy (SEM) for morphological changes, colony formation and wound healing assays for proliferation and migration, and flow cytometry for cell cycle and apoptosis analysis. Gene expression changes were evaluated using a qRT-PCR array targeting 84 genes involved in cancer progression. PFDN1 silencing resulted in a 54.8% reduction in PFDN1 protein levels (p < 0.0001). Morphological analysis revealed cytoplasmic shrinkage, chromatin condensation, roughened membranes, and microvilli loss, consistent with apoptotic changes. Colony formation assays showed a 10.33% reduction in colony number and size (p < 0.05) in PFDN1-silenced cells. Migration was significantly impaired, with reduced wound closure observed in wound healing assays (p < 0.01). Flow cytometry revealed a G2/M phase arrest (p < 0.05) and increased early apoptotic populations (20.93% vs. 5.42% in controls, p < 0.01). Gene expression analysis showed downregulation of genes associated with angiogenesis (KDR, TEK), EMT (FOXC2, SNAI1), and hypoxia signaling (CA9, EPO), while proapoptotic genes, such as FASLG, were upregulated. This study highlights the critical role of PFDN1 in TNBC progression, demonstrating that its silencing disrupts survival, migration, cell cycling, and apoptosis pathways. PFDN1 knockdown also significantly alters the expression of key cancer-related genes, further impairing angiogenesis, EMT, and hypoxia adaptation. These findings suggest that targeting PFDN1 could be a promising therapeutic strategy for TNBC, warranting further investigation in preclinical models. Show less

Natural history studies of pediatric rare neurometabolic diseases are important to understand disease pathophysiology and to inform clinical trial outcome measures. Some data collections require sedation given participants' age and neurocognitive impairment. To evaluate the safety of sedation for research procedures, we reviewed medical records between April 2017 and October 2019 from a natural history study for CLN3 (NCT03307304) and one for GM1 gangliosidosis (NCT00029965). Twenty-two CLN3 individuals underwent 28 anesthetic events (age median 11.0, IQR 8.4-15.3 years). Fifteen GM1 individuals had 19 anesthetic events (9.8, 7.1-14.7). All participants had the American Society of Anesthesiology classification of II (8/47) or III (39/47). Mean sedation durations were 186 (SD = 54; CLN3) and 291 (SD = 33; GM1) min. Individuals with GM1 (6/19, 31%) were more frequently prospectively intubated for sedation (CLN3 3/28, 11%). Minor adverse events associated with sedation occurred in 8/28 (28%, CLN3) and 6/19 (32%, GM1) individuals, frequencies within previously reported ranges. No major adverse clinical outcomes occurred in 47 anesthetic events in pediatric participants with either CLN3 or GM1 gangliosidosis undergoing research procedures. Sedation of pediatric individuals with rare neurometabolic diseases for research procedures is safe and allows for the collection of data integral to furthering their understanding and treatment. Show less